Membrane Transport by Guinea Pig Peritoneal Exudate Leukocytes: Effect of Phagocytosis on Hexose and Amino Acid Transport I t * DAVID C. STRAUS 3.4 JOHN G. IMHOFF AND PETER F. BONVENTRE Department of Microbwlogy, University of Cincinnati College of Medicine, Cincinnati Ohio 45267

ABSTRACT Short term, carrier mediated transport of D-glucose, L-leucine and L-lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two-limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a K, of 0.1 mM for L-leucine and 0.05 mM for L-lysine; Vmaxvalues were 2.0 and 2.85 nmolelmg protein190 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat-killed staphylococci by phagocytosis stimulated 2-deoxy-Dglucose (2-dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2-dOG increased in post-phagocytic macrophages, the kinetics of the system were not altered significantly. The K, for both pre- and post-phagocytic transport of 2-dOG was shown to be 1.2 mM and the Vmaxwas shown to increase from a pre-phagocytic value of 20 nmoleslmg protein190 seconds to a post-phagocytic 27 nmoleslmg protein190 seconds. Phagocytosis of heat-killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages. Membrane transport of sugars and amino acids in phagocytic cells is accomplished by carrier mediated facilitated diffusion (Gee et al., '71; Bonventre and Mukkada, '74; Mukkada and Bonventre, '74) as is the case in most mammalian cells (Rothstein, '68); exceptions include kidney tubule and intestinal epithelial cells which, like prokaryotic cells, utilize a mechanism of active transport (Elbrink and Bihler, '75). Our studies have established characteristics of glucose transport in mouse and guinea pig peritoneal macrophages (Mukkada and Bonventre, '74) and have shown, in addition, that phagocytic stimulus results in augmentation of glucose transport by these cells (Bonventre and Mukkada, '74). The degree to which the increase in hexose J. CELL. PHYSIOL., 93: 105-116.

uptake following phagocytosis by macrophages can be ascribed to changes in the glucose transport system per se &e., changes in number or affinity of transport sites) will be the subject of another publication (Straus et al., '77). In the present study, we extend the previous observations on the post-phagocytic stimulation of glucose transport by guinea pig peritoneal macrophages and characterize the kinetics of leucine and lysine transport by the Received Mar. 3, '77. Accepted Apr. 5, '77. ' Presented, in part, at the 1975 Annual Meeting of the American Society for Microbiology in New York. *Supported by grant DA-ARO-D-31-124-73-G123from the Life Sciences Division, U. S. Army Research Office, Department of the Army. Present address: Department of Microbiology. The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 (512-696-6506). Person to whom correspondence should be addressed.

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D. C. STRAUS, J. G. IMHOFF AND P. F. BONVENTRE

same cell type. In addition, an attempt is made to reconcile the data with that of Tsan and Berlin ('71b) who studied amino acid transport in phagocytic cells and advanced the hypothesis that membrane functions of phagocytic cells are topographically separated and, under appropriate conditions, that amino acid transport sites on the plasma membrane are preserved during phagocytosis. MATERIALS AND METHODS

PeritoneaE exudate cells (PEC) Peritoneal macrophages were obtained from Hartley strain guinea pigs (Camm Research Institute, Wayne, New Jersey). Macrophage cultures were established as previously described (Bonventre and Mukkada, '74). Peritoneal exudate cells were allowed to attach to glass coverslips for two hours in NCTC-135 (Gibco, Grand Island, New York) plus 20% fresh guinea pig serum in a n environment of 5% CO, a t 37°C. Cultures were washed once in pre-warmed Hanks Balanced Salt Solution (HBSS) to remove nonadherent cells. The adherent cell population was then incubated in NCTC plus 5%fresh guinea pig serum for another two hours. More than 95% of the adherent cell populations were judged to be mononuclear phagocytes by microscopic evaluation of Giesma-stained preparations. Peritoneal polymorphonuclear leukocytes were also obtained from Hartley strain guinea pigs (Camm Research Institute, Wayne, New Jersey). PMN coverslip cultures were established from guinea pig peritoneal exudates harvested three hours earlier with 20 ml of 4% beef heart infusion as previously described (Bonventre and Mukkada, '74). After the peritoneal exudate cells were allowed to attach for one hour in NCTC plus 20% fresh guinea pig serum a t 37"C, the coverslip cultures were washed once in warm HBSS to remove nonadherent cells. More than 95%of the adherent cells were judged to be polymorphonuclear phagocytes by microscopic evaluation of Giesma-stained preparations.

brated in 5% C02-air a t 37°C before use. Staphylococci were used a t a concentration of lo8 organisms/ml and latex spheres a t a concentration of 0.5% (v/v). Phagocytosis was allowed to proceed under the same conditions as for culture. Uptake was monitored by phase contrast microscopy. The time allowed for phagocytosis of latex particles and killed staphylococci was 30 minutes and 1 hour respectively. After phagocytosis, the cells were rinsed in HBSS and processed as previously described (Bonventre and Mukkada, '74).

Glucose uptake After removal of the tissue culture fluids or phagocytic menstruum by aspiration, coverslips were rinsed three times with glucosefree HBSS (prewarmed to 37"C), and then incubated with glucose-free HBSS for 30 minutes a t 37°C to deplete endogenous hexoses. The fluids were then removed by aspiration and the coverslips were drained and placed on a warming tray, preheated to 38.5"C, with one edge overhanging so they could be easily grippled with forceps (Hawkins and Berlin, '69). Exactly 350 pl of prewarmed glucose-free HBSS containing P4CI 2deoxy-D-glucose(New England Nuclear Corp., Boston, Massachusetts), (in concentrations ranging from 0.10 mM to 4.0 mM specific activity of 0.2 pCi/pmole) were then placed over the monolayer. After incubation for 90 seconds the coverslips were rinsed thoroughly by dipping serially in four beakers containing cold glucose-free HBSS. Hawkins and Berlin ('69) have shown that loss of intracellular label due to diffusion during such a rinsing procedure is negligible. Coverslips were allowed to air dry, broken into several pieces, then dropped into scintillation vials containing 10 ml Bray's scintillation fluid (Bray, '60). Radioactivity was measured in a Packard liquid scintillation spectrophotometer (Packard Instrument Co., La Grange, Illinois). Results were calculated as nanomoles 2-deoxy-D-glucose per mg cell protein per 90 seconds (nm 2dOG/mg/90 seconds). Six samples were taken Phagocytosis a t each recorded interval. Control cell culTo evaluate the effect of phagocytosis on tures were run a t 4°C to measure non-specific glucose and amino acid transport, two par- adherence of 2-dOG to the cells or particles ticles were employed: (i) polystyrene latex not removed by the rinsing procedure. spheres, 1.2 p m in diameter (Dow Chemical Protein concentrations (both pre- and postCo.); or (ii) a heat-killed suspension of S. phagocytosis) were measured by the method aureus 502A. The particles were suspended in of Oyama and Eagle ('56). The protein content NCTC plus 10%guinea pig serum and equili- of six coverslip monolayers was determined

107

TRANSPORT BY GUINEA PIG PHAGOCYTES

-

and the values were averaged for use in the calculations of glucose transport. Amino acid uptake Coverslip monolayers were prepared and handled in the same way as those employed in measurements of glucose transport. To prevent potential incorporation of amino acids into cellular proteins, diphtheria toxin a t a concentration of 50 MLD/ml was added to the coverslip culture fluids for two hours to shut down protein synthesis before uptake experiments were initiated. Diphtheria toxin a t this concentration has not been shown to influence amino acid transport in these cells (unpublished results). After cell monolayers had been rinsed thoroughly t o remove traces of toxin, 350 p l of prewarmed HBSS containing [14C1-lysine (uniformly labelled) or P4CI-leurine (uniformly labelled; New England Nuclea r Corp., Boston, Massachusetts) in concentrations ranging from 0.025 mM to 0.20 mM (specific activity of 2 pCi/umole) were then placed on the monolayers as described previously. Uptake of amino acids was allowed to proceed for 90 seconds at 385°C. Calculated values represent initial velocity of transport since uptake was shown to be linear for a t least 90 seconds. The concentration of “cold” amino acids used in the inhibition experiments with P4C1 L-leucine was 2.0 mM. Values were corrected for non-specific adherence of label by subtracting trace amounts measurable in samples incubated at 4°C. Treatment with colchicine Where appropriate, after the 4-hour treatment with NCTC plus fresh guinea pig serum, the cells were exposed to differeent compounds a t varying concentrations to determine their effect on glucose andlor amino acid transport. Colchicine (Nutritional Biochemical Corporation, Cleveland, Ohio) was used in M to concentrations ranging from 5 x low6M with an exposure time of 30 minutes. Colchicine was diluted in 2% fresh guinea pig serum and NCTC. In experiments where colchicine and phagocytosis were involved, the same concentration of colchicine used in the pretreatment step was present in the phagocytic media for the duration of the experiment. Calculations The net uptake of amino acids and 2-deoxyD-glucose is expressed as nanomoles per mg of

cell protein 90 seconds (nm/mg/90). Data were calculated from the average radioactivity per coverslip, average protein content per coverslip, and the calculated specific activity of the uptake fluid. Each datum point represents an average of six independent observations. All initial velocity uptakes were based on 90-second incubation periods. Corrections for diffusion was calculated by the method of Akedo and Christensen (’62) using the assumption that cell volume is directly related to cell protein (Groth and Rosenberg, ’72). The corrected initial velocity values were plotted by the double reciprocal method of Lineweaver and Burk (’34) to determine apparent Michaelis constant (K,) and apparent maximal uptake velocities (Vmax).The inhibition constant (K,) which characterizes the affinity of an amino acid or an amino acid analogue for its carrier on the plasma membrane, was determined by measuring the compound’s capacity to inhibit leucine transport. In this survey, the concentration of L-leucine used was its apparent K, (i.e., 0.1 mM). The K, of inhibitor was calculated from the forv -I mula K, = -as described by Tsan and 2v - v,

Berlin (’71a). [I1 is the concentration of inhibitor and V, and V are the velocities in the presence and absence of inhibitor, respectively. RESULTS

Saturation kinetics Uptake of L-lysine and glucose by cultured guinea pig peritoneal macrophages were shown to be saturable processes. Figure 1 shows that at concentrations of L-lysine between 20 mM to 50 mM, the ratio of uptake (V) divided its medium concentration [SI to the reciprocal of its concentration [l/Sl decreased linearity with increasing amino acid concentration. The apparent diffusion constant (KD) claculated from the resulting straight line, according to the procedure of Akedo and Christensen (’62), was 0.185 pliter/mg protein/90 sec. This represents less than 1%of the amino acid normally transported during these experiments. Calculations of initial uptake velocities were corrected for diffusion in all cases. The kinetics of leucine and lysine transport The initial velocities of leucine and lysine uptake were measured at substrate concentrations of 0.050-8.0 mM. The calculated up-

108

D. C. STRAUS, J. G. IMHOFF AND P. F. BONVENTRE 3 -I

'1

I

01

I

d2 VS (mhl-')

.d3

.04

.05

Fig. 1 Plot of L-lysine uptake (V) by guinea pig peritoneal macrophages divided by the lysine concentration (S) versus the reciprocal of 6). The apparent diffusion constant (KD) calculated from the ordinate intercept was 0.185r l per mg of protein in 90 seconds. Results represent the average uptake of six coverslips a t each datum point. In all cases uptake was measured on the day PEC were established in in vitro cultures.

zl rn 0

T

1.0-

take velocities were corrected for diffusion and converted to nmoles/mg protein/90 seconds. The data presented in double reciprocal plots (figs. 2, 3) show that the apparent K, and V,,, values for both leucine and lysine transport and these results were shown to be reproducible from experiment to experiment. A distinct two-limbed curve was obtained

with a sharp break in linearity a t an amino acid concentration of approximately 0.4 mM. This could indicate two distinct transport systems in these cells or perhaps merely changes in leucine and lysine-binding proteins at higher concentrations in these facilitated diffusion systems. Two-limbed double reciprocal plots have been described in some mamma-

109

TRANSPORT BY GUINEA PIG PHAGOCYTES

u

-20 -le -16 -14 42 -10 -6 -6 -4 -2 0 2 4 I/S (mM-')

6

8

10 12 14

16 I8 20

Fig. 3 Initial velocity of facilitated lysine transport by normal guinea pig peritoneal macrophages grown on coverslips. Cell monolayers were incubated in the presence of [I4C1lysine in concentrations from 0.050 to 8.0 mM. The monolayers were then rinsed and counted as described. All points represent an average of six determinations. In all cases, uptake was measured on the day PEC were established in in vitro cultures.

v)

4'0

1

I/S (mM'') Fig. 4 Initial velocity of facilitated leucine transport by normal guinea pig peritoneal macrophages grown on coverslips. Legend same as figure 2 except cell monolayers were incubated in the presence of I"C1-leucine in concentrations from 0.025 mM to 0.20mM. In all cases, uptake was measured on the day PEC were established in in vitro cultures.

lian cell systems for transport of amino acids: isolated rabbit renal tubules for L-proline (Hillman and Rosenberg, '69), for glycine in the Same system (Hillman et al., '68), for lysine, arginine and tryptophan in human fibroblasts (Groth and Rosenberg, '72) and for L-isoleucine in cultured human fibroblasts (Hillman and Otto, '74). Although the presence of two apparent K, values for each amino acid does not conclusively prove the ex-

istence of two distinct transport systems for each, the possibility cannot be excluded.

Examination ofthe low concentration limb of amino acid transport Because both amino acid transport system displayed K,'s approximately 0.1 mM and 5.0 mM, it is probable that the lower K, represents the activity of the physiologically s i g nificant transport system, however, this de-

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D. C. STRAUS, J. G . IMHOFF AND P. F. BONVENTRE

I/S (mM-')

Fig. 5 Initial velocity of facilitated lysine transport by normal guinea pig peritoneal macrophages grown on coverslips. Legend same as figure 3 except cell monolayers were incubated in the presence of ["Cl lysine in concentrations from 0.025 mM to 0.20 mM. In all cases, uptake was measured on the day PEC were established in in vitro cultures. TABLE 1

Effectofcolchicine M) duringphagocytosis on membrane transport ofleucine and lysine by guinea pig peritoneal macrophages. All experiments were performed at 0.I mM amino acid concentrations. All initial uptake velocities were recorded as nanomoles/mg proteid90 seconds. Each value represents an average of six determinations Colchicine

Lysine transport Leucine transport

+

Normal

+ Colchicine

+ Phagocytosis

phagocytosis

2.14 0.91

1.90 0.89

2.04

2.10 0.92

0.86

'

The macrophages were exposed to heat-killed staphylococci a t a concentration of lo8 organismslml for one hour. 'This concentration of colchicine was not seen to inhibit phagocytosis.

pends on the actual concentration of amino acid that is normally present in this environment. Initial uptake velocities were once again calculated over an amino concentration range in the incubation medium of 0.025-0.2 mM, and the data are shown in figures 4 and 5. The derived values of K, and V,, of both leucine and lysine transport were shown to be constant. As shown in figure 4, the low concentration limb of the leucine curve established a K, of 0.1 mM and a V,, of 2.0 nmoleslmg proteini90 seconds. In figure 5, the low concentration limb of the lysine curved was seen t o establish a K, of 0.05 mM and a V,, of 2.85 nmoles/mg protein190 seconds.

Effectsof cell modifiers The effects of cell modifiers on amino acid uptake were studied at one concentration point on the low concentration limb (0.1 mM). Colchicine (5 x M M) treatment of guinea pig peritoneal macrophages for 30

minutes did not inhibit leucine or lysine uptake (table 1) and did not cause cell death (as determined by neutral red and tyrpan blue staining) or any loss of cellular protein, as determined by protein analysis (Oyama and Eagle, '56). Phagocytosis by these cells of either latex beads or heat-killed staphylococci did not alter amino acid transport. Also phagocytosis in the presence of colchicine MI, after a 30-minute pre(5x10-5 to treatment with colchicine, did not decrease amino acid transport in this cell system.

Effects of competitive inhibition Competitive inhibition studies of leucine uptake in the presence of 2 mM lysine demonstrated that under these conditions leucine transport was decreased from 50-60% A double reciprocal plot of these data show that lysine is indeed a competitive inhibitor of leucine uptake (fig. 6). In the work of Tsan and Berlin ('71a), leucine completely inhib-

111

TRANSPORT BY GUINEA PIG PHAGOCYTES 4.0-

I -40

I -20

I

I

I

-10

5

10

I/S

1

I 20

40

(mM-')

Fig. 6 Inhibition of leucine transport in normal guinea pig peritoneal macrophages by 2 mM lysine. Cell monolayers were incubated with various concentrations of i"CI leucine for 90 seconds in the presence of the inhibitor. Each point represents the average of six determinations. In all cases uptake was measured on the day PEC were established in in vitro cultures. O---O,normal leucine transport; L O , leucine transport in the presence of 2 mM lysine.

ited the transport of lysine in the rabbit alveolar macrophage. Tsan was able to demonstrate over 90%inhibition of lysine transport. Our cell system under these conditions showed about 65%inhibition. Because figure 6 demonstrates simple competitive inhibition, i t is probable that if the lysine concentration was raised high enough, the inhibition of leucine transport by lysine would reach the figure reported by Tsan. Other experiments demonstrated similar results for inhibition of lysine transport by leucine (data not shown). The Kis which characterize the affinity of other amino acids to the leucine carrier may be easily calculated by the formula listed in the MATERIALS AND METHODS section. The results obtained by using eight amino acids and 0.1 mM leucine are shown in table 2. Of the eight amino acids tested, only the basic amino acids, lysine, arginine, tryptophan and histidine, show any consequential affinity for the leucine carrier. Isoleucine, a neutral amino acid, was shown to have a fairly high affinity. The other amino acids tested, glutamic acid, glycine and valine, were either neutral or acidic in nature and did not compete SUCcessfully for the leucine carrier site.

Effect of phagocytosis on 2-deoxy-D-glucose uptake in guinea pig peritoneal macrophages After phagocytosis of foreign particles, the uptake of 2-deoxy-D-glucosewas greater than

TABLE 2

Inhibition of leucine transport in guinea pig peritoneal macrophages. Cell monolayers were incubated with 0.1 mM P4CI leucine in the presence of the inhibitor Amino acids Inhibitor

L-lysine L-arginine L-tryptophan L-isoleucine L-histidine L-valine L-glycine L-glutamic acid

Ki (mM1

0.72 1.84 2.18 2.73 3.30 8.46 > 10.00 > 10.00

Cell inhibitors were used at 2 mM concentrations The amount of inhibition was determined by comparison with a control without inhibitor The K, was calculated from the equation listed in the MATERIALS AND METHODS section Each value represents an average of six determinations

in macrophages that had not undergone phagocytosis. This was true for heat-killed staphylococci ingestion, as well as the ingestion of latex particles. The level of increase of hexose uptake was usually found to be between 30 and 50%. All uptake experiments were performed for 90 seconds instead of the 15 to 20 minute incubation periods previously used (Bonventre and Mukkada, '74; Mukkada and Bonventre, '74). The rate of 2-deoxy-Dglucose uptake was determined a t substrate concentrations of between 0.25 and 4.0 mM. Controls were run in the cold (4°C) to check for nonspecific "transport" due to tracer 2-

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D. C. STRAUS. J. G. IMHOFF AND P. F. BONVENTRE

dOG adhering or localizing in crevices formed by phagocytic particles that were not removed during the vigorous washing procedure. These controls showed approximately 2-4%non-specific uptake due to incomplete washing of the monolayer. The data were calculated in terms of nanomolesfmg proteinf90 seconds and expressed in the Lineweaver-Burk plot (fig. 7). Ingestion of heat-killed staphylococci normally resulted in an increase of cellular protein of 2-5% after appropriate washing procedures were performed to remove any adhering bacteria. These values were taken into consideration when the amount of 2-dOG transported by these cells was calculated. Although the rate of hexose uptake was seen to increase after particle ingestion, the apparent K, did not change significantly (from 1.2 to 1.1mM). However, an increase in the rate of hexose upwhich intake was reflected in the V,,, creased from 20.0 nmoles to 26.6 nmoles after particle ingestion. Only one transport system for 2-deoxy-D-glucosein guinea pig peritoneal macrophages could be resolved from these data.

.

-2 -I I/S(rnM)

0

2

3

4

5

Fig. 7 Lineweaver-Burk plot of hexose transport in preand post-phagocytic guinea pig macrophages. Uptake of 2deoxy-D-glucose was determined as described in MATERIALS AND METHODS; the concentrations of hexose ranged from 0.25 to 4.0 mM and uptake was followed for 90 seconds. Each point represents the average of six determinations. In all cases uptake was measured on the day PEC were established in in vitro cultures. G O ,non-phagocytosing cells; 0-0, post-phagocytic cells; heat killed S. aureus 502A (lonorganisms/mlf incubated with PEC for one hour.

,

/,

- 2 - 1

I

0

I

2

3

4

5

I/SbnM)

Fig. 8 Lineweaver-Burk plot of hexose transport in preand post-phagocytic guinea pig peritoneal polymorphonuclear leukocytes. Uptake of 2-deoxy-D-glucose was determined as described in MATERIALS A N D METHODS: the concentration of hexose ranged from 0.25 to 4.0 mM and uptake was followed for 90 seconds. Each point represents the average of six determinations. I n all cases uptake was measured on the day PEC were established in in vitro cultures. Legend same as for figure 8.

Effect of phagocytosis on 2-deoxy-D-glucose uptake in guinea pig PMNs The tranport of 2-dOG in the post-phagocytic polymorphonuclear leukocyte was also examined. All experiments involved phagocytosis of heat-killed staphylococci. The increase in cellular protein in the post-phagocytic PMN as opposed to the normal PMN was similar to that observed for the guinea pig macrophage (see above). When compared to the post-phagocytic guinea pig macrophage, the post-phagocytic guinea pig PMN did not transport significantly increased quantities of hexose after particle ingestion. The values obtained for the rate of increase of hexose transport in the post-phagocytic PMN fell within a range of between 0 and 14%with most of the values less than 4%. A typical LineweaverBurk plot of this data is presented in figure 8. The K,,, and V,,, for the pre-phagocytic PMN were 0.75 mM and 28.6 nmolesfmg protein190 seconds, respectively. These values did not change significantly in the post-phagocytic guinea pig polymorphonuclear leukocyte. DISCUSSION

Guinea pig peritoneal macrophages transport glucose by facilitated diffusion (Rothstein, '68). [U-'*C-I 2 deoxy-D-glucose has been shown to share the same transport car-

TRANSPORT BY GUINEA PIG PHAGOCYTES

rier a s glucose in the mononuclear phagocyte (Mukkada and Bonventre, '74). Previous reports from this laboratory have established t h a t after phagocytosis, the total quantity of glucose taken into the macrophage before saturation increases significantly. I t also appeared that the kinetics of transport were altered after the phagocytic stimulus, suggesting that the K, value for glucose transport was reduced indicating either a change in the affinity of hexose transport sites on the plasma membrane for substrate or alternatively an increase in the number of sites following phagocytosis (Mukkada and Bonventre, '74; Bonventre and Mukkada, '74). These hypotheses were based on calculations made from values of glucose uptake determined after 10 or 15 minutes of incubation with 2 mM 2-deoxy-D-glucose. Since those reports appeared, we have established that linearity of glucose uptake by these cells is maintained for only approximately two minutes, after which time the rate of uptake decreases incrementally until saturation levels are achieved. This made it clear that our previous analysis of transport kinetics required reevaluation with data obtained during short periods of hexose uptake (i.e., less than 2 minutes). Thus, all experiments described in this report are based on values of 2 dOG uptake measured after 90 seconds of incubation. The amount of sugar in the cell expressed as nM/mg proteid90 seconds thus represents a more accurate measure of initial rates of transport velocity than was obtained previously. The data based on these short-term uptake measurements show that phagocytosis does indeed induce a n increase in the uptake of 2 deoxy-D-glucoseby guinea pig peritoneal macrophages but that the apparent K, value does not change appreciably. The V,,, value, however, does increase after phagocytosis. We conclude that the initial interpretation of data based on longterm uptake values is incorrect (Bonventre and Mukkada, '74). Rather, i t now appears that the number of transport sites may increase but that the affinity of hexose for the transport sites on the membrane is unaltered after phagocytosis. This situation is analogous to the increased hexose transport known to be associated with virus induced cell transformation reported by Weber ('73). There, transport of 3-0-methylglucose (a nonphosphorylatable glucose analogue which is also transported by the glucose system) was four to five times faster in trans-

113

formed chick embryo fibroblasts than in normal chick embryo fibroblasts growing a t the same rate. Kinetic measurements revealed that K,'s for transport of 3-0-methyl-glucose or of 2-deoxy-D-glucose were not altered significantly by viral transformation of those for hexose transport cells. Only the V,, showed any consistent, significant alteration. It was suggested that an increased V,,, value could result either by synthesis of an increased number of transport sites per cell as hypothesized by Isselbacher ('721, or by unmasking allosteric modification of preexisting sites, as proposed by Roseman ('69). Data in the present study show that guinea pig macrophages possess the capacity to sequester excess hexose which might be required for increased energy demands associated with phagocytosis. Whether or not phagocytosis entails a significant increase in the glucose requirement is unclear. The observations of West et al. ('68) suggest that the phagocytizing macrophage does not utilize exogenous glucose to a greater extent than does the resting phagocyte; the data show that exogenously supplied glucose is utilized to the same extent by mononuclear phagocytes when in the resting state or following a phagocytic stimulus. Thus one must assume that either additonal energy demands associated with phagocytosis are inconsequential, or alternatively, that energy requirements are met by intracellular glycogen stores present in mononuclear phagocytes. In the latter regard, it is pertinent that inflammatory mononuclear cells contain considerable quantities of cytoplasmic glycogen (Gudewicz and Filkins, '76). We have recently shown (Bonventre et al., '77) that immunological activation of macrophages also results in marked augmentation of hexose transport. In those studies initial uptake velocities were measured (90 seconds) so there was no doubt that transport rather than channeling of nutrients into cellular components was being reflected by the values. An increase in glucose uptake following activation of macrophages was demonstrated with peritoneal cells of mice and guinea pigs sensitized by infection with three different bacterial pathogens. Examination of this phenomenon of a u g mentation of hexose transport in phagocytic cells after particle ingestion was then extended to polymorphonuclear leukocytes. Augmentation did not appear to be present in

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D. C. STRAUS, J. G . IMHOFF AND P. F. BONVENTRE

post-phagocytic guinea pigs peritoneal PMNs (fig. 8).The usual increase in 2-dOG transport after phagocytosis by PMNs was approximately 2-5%. These values are probably not significant and a closer examination of the kinetics of the 2-dOG transport system in these cells, as compared to the peritoneal macrophage, suggests an explanation of why augmentation of hexose transport is absent in post-phagocytic polymorphonuclear leukocytes. Guinea pig peritoneal PMN normally transport hexose a t a rate approximately 1.5 times as fast as the normal peritoneal macroin both the normal guinea phages. The V,, pig peritoneal PMNs and post-phagocytic guinea pig peritoneal macrophages are practically the same, 28.6 nmoledmg prot/90 seconds and 27 nmoledmg prot/90 seconds, respectively. Phagocytosis of foreign particles by the PMN did not alter either the K, or the V,, of its hexose transport system. This indicates that the normal peritoneal PMNs transport as much hexose as do post-phagocytic peritoneal macrophages. It would appear then that the various types of phagocytic cells appear to respond differently to phagocytic stimuli, and caution should be exercised in generalizing results based on these kinds of experiments when only one cell type is examined. The observation that hexose transport is not decreased after particle ingestion also suggests that glucose transport sites are not internalized during phagocytosis. The phagocytosis of large numbers of foreign particles results in the internalization of large amounts of plasma cell membrane (Tsan and Berlin, ’71b). The phagocyte must be able to continue carrier-facilitated transport of many vital compounds after this substantial loss of membrane surface area, if it is to obtain the nutrients required to carry out its normal and perhaps augmented metabolic functions. To this end, the phagocyte must either synthesize new receptor sites as quickly as the old one are being internalized, or retain specific old receptor sites on the cell surfaces as phagocytosis occurs. The latter possibility would of course necessitate a topographical separation of unique sites involved in either transport or phagocytosis. Evidence for a topographical separation of sites of a mosaic nature for the macrophage membrane comes from the observation by Griffin and Silverstain (‘76) that ingestion of one particle does not trigger generalized phagocytosis of

all particles attached to the cell membrane. The authors suggested that the order to phagocytize is received only in the segment of the cell membrane immediately adjacent to the phagocytized particle. Further evidence for a topographical separation of transport and phagocytosis sites was obtained by Tsan and Berlin (’71b), who used rabbit alveolar macrophages to demonstrate t h a t lysine transport sites were not rapidly resynthesized after phagocytosis but were preserved on the cell surface. Our results corroborate the results of Tsan and Berlin (‘71b), who suggested that amino acid transport sites are preserved following phagocytosis of particles by guinea pig peritoneal macrophages, thus supporting the topographical separation of membrane function hypothesis. Table 1 shows, however, in the case of guinea pig macrophages, colchicine treatment and phagocytosis in the presence of colchicine does not cause any measurable reduction in amino acid transport as reported by Ukena and Berlin (‘72) for the rabbit polymorphonuclear leukocyte. Although in disagreement with the report of Ukena and Berlin (‘721, our data regarding the effect of treatment with colchicine on amino acid transport in post-phagocytic leukocytes do correlate with the results of Dunham et al. (‘74). These workers found that colchicine disruption of microtubules had no effect on the amino acid transport systems of human peripheral PMNs and no effect on inhibition of membrane transport after exposure to various phagocytic stimuli. Dunham and co-workers (’74) attribute these differences to the fact that different types of phagocytic cells were used, as well as cells from different species. Also, in the study by Dunham et al. (‘74), cells in suspension were used, whereas Berlin’s group used cells that had been allowed to adhere to glass. Dunham et al. (’74) suggested that “attachment to glass may be associated with changes in the functional state of the PMNs.” These results and the data presented herein indicate that the effect of colchicine on the amino acid transport of the post-phagocytic leukocyte depends on many variables. Guinea pig peritoneal macrophages were shown to possess two distinct transport systems for leucine and lysine (figs. 2,3). The low concentration limb K, for leucine in these cells was 0.1 mM (fig. 4) and the K, for lysine was 0.05 mM (fig. 5). Both of these values

TRANSPORT BY GUINEA PIG PHAGOCYTES

agree well with the K, of 0.1 mM reported by Tsan and Berlin ('71a) for rabbit aveolar macrophages. The calculated Ki's for the eight different amino acid inhibitors (table 2 ) of leucine transport gave further information about the nature of these transport systems. Of the amino acids examined, L-lysine had the highest affinity for the carrier with a K, of 0.72. Other basic amino acids such as arginine and histidine (unsaturated aromatic amino acid) also showed strong affinities for the carrier, indicating that an imidazole or guanido group a t the end of the side chain (terminal positive charge) increases the affinity for carrier. Isoleucine also showed an affinity for the carrier, which was in all probability due to its structural similarity to leucine. The structural basis for the affinity of the other unsaturated amino acid, tryptophan, is unclear. Neutral amino acids (valine and glycine) and an amino acid with a carboxylic group a t the end of its side chain (glutamic acid) were seen to have little or no affinity for the carrier. The results presented in table 2 follow the general pattern of amino acid analogue inhibitors of the AM-I system described by Tsan and Berlin ('71a). However, the affinities reported here are not as high as those reported in the AM-I system. LITERATURE CITED Akedo, H., and H. N. Christensen 1962 Nature of insulin action on amino acid uptake by t he isolated diaphragm. J. Biol. Chem., 237: 188-222. Bonventre, P. F., and A. J. Mukkada 1974 Augmentation of glucose transport in macrophages after particle ingestion. Infect. Immunity, 10: 1391.1396. Bonventre, P. F., D. C. Straus, R. E. Baughn and J. G. Imhoff 1977 Enhancement of carrier mediated transport after immunological activation of peritoneal macrophages. J. Immunol., 118: 1827-1835. Bray, G. A. 1960 A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem., 1: 279-285. Dunham, P. B., I. M. Goldstein,and G. Weissman 1974 Potassium and amino acid transport in human leukocytes exposed to phagocytic stimuli. J. Cell. Biol., 63: 215-226. Elbrink, J., and I. Bihler 1975 Membrane transport: Its relation to cellular metabolic rates. Science, 188: 11771184. Gee, J. B. L., A. S. Khadndwala and R. W. Bell 1974 Hexose transport in the alveolar macrophage: Kinetics and pharmacologic features. J. Reticuloendothel. SOC.,15: 394405.

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Griffin, F. M., and S. C. Silverstain 1974 Segmental response of the macrophage plasma membrane to a phagocytic stimulus. J. Exp. Med., 139: 323-336. Groth, U., and L. E. Rosenberg 1972 Transport of dibasic amino acids, cystine, and tryptophan by cultured human fibroblasts: Absence of a defect in cystinuria and bartnup disease. J. Clin. Invest., 51: 2130-2142. Gudewicz, P. W., and J. Filkins 1976 Glycogen metabolism in inflammatory macrophages. J. Reticuloendothelial SOC., 20: 147-152. Hawkins, R. A,, and R. D. Berlin 1969 Purine transport in polymorphonuclear leukocytes. Biochem. Biophys: Acta., 173: 324-337. Hillman, R. E., I. Albrecht and L. E. Rosenberg 1968 Identification and analysis of multiple glycine transport systems in isolated mammalian renal tubules. J. Biol. Chem., 243: 5566-5571. Hillman R. E., and L. E. Rosenberg, 1969 Amino acid transport by isolated mammalian renal tubules. 11. Transport systems for L-proline. J. Biol. Chem., 244: 4494-4498. Hillman, R. E., and E. F. Otto 1974 Transport of L-isoleucine by cultured human fibroblasts; uptake by normal cell lines and isolation of a cell line lacking sodium dependent uptake. J. Biol. Chem., 249: 3430.3435. Isselbacher, K. J. 1972 Increased uptake amino acids and 2-deoxy-D-glucoseby virus-transformed cells in culture. Proc. Nat. Acad. Sci. (U. S. A,), 69: 585-589. Lineweaver, H., and D. Burk 1934 The determination of enzyme dissociation constants. J. Amer. Chem. SOC., 56: 658-666. Mukkada, A. J., and P. F. Bonventre 1974 Membrane transport by mouse and guinea pig macrophages: Characteristics of the glucose transport system. J Reticuloendothel. SOC.,17: 20-29. Oyama, V. I., and H. Eagle 1956 Measurement of cell growth in tissue culture with a phenol reagent (FolinCiocolteau). Proc. SOC. Exp. Biol. Med., 91: 305-307. Roseman, S. 1969 The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol., 54: 1385.1845. Rothstein, A. 1968 Membrane phenomena. Annu. Rev. Physiol., 30: 15-72. Straus, D. C., J. G. Imhoff and P. F. Bonventre 1977 Membrane transport of amino acids and hexose by guinea pig and mouse phagocytes. J. Reticuloendothel. Soc., in press. Tsan, M. F.,and R. D. Berlin 1971a Membrane transport in the rabbit alveolar macrophage. The specificity and characteristics of amino acid transport system. Biochim. Biophys. Acta., 241: 155-169. Tsan, M. F. ,and R. D. Berlin 1971b Effect of phagocytosis on membrane transport of nonelectrolytes. J. Exp. Med., 134: 1016-1035. Ukena, T. E., and R. D. Berlin 1972 Effect of colcbicine and vinblastine on the topographical separation of membrane function. J. Exp. Med., 136: 1-7. Weber, M. J. 1973 Hexose transport in normal and in Rous Sarcoma Virus-transformed cells. J. Biol. Chem., 248: 2978-2983. West, J., D. J. Morton, V. Esmann and R. L. Stjernholm 1968 Carbohydrate metabolism in leukocytes. Arch. Biochem. Biophys., 124: 85-90.

Membrane transport by guinea pig peritoneal exudate leukocytes: effect of phagocytosis on hexose and amino acid transport.

Membrane Transport by Guinea Pig Peritoneal Exudate Leukocytes: Effect of Phagocytosis on Hexose and Amino Acid Transport I t * DAVID C. STRAUS 3.4 JO...
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